The objectives of the proposed research are to develop further an alphavirus replicon particle vaccine against human cytomegalovirus (CMV). CMV causes a chronic, persistent infection and is an important cause of morbidity and mortality in congenitally infected children and in individuals with impaired immune systems such as transplant recipients. No vaccine is currently available to prevent CMV disease, which is responsible for many billions of dollars in annual healthcare costs. We propose a novel approach, using a propagation-defective, single cycle, RNA replicon vector system, derived from an attenuated strain of Venezuelan equine encephalitis (VEE) virus, to produce virus-like replicon particles (VRP) expressing the genes for three CMV proteins (pp65, IE1 and gB) that have been identified as the targets of protective immunity. Preliminary animal studies have demonstrated the feasibility of this approach, as VRP expressing CMV proteins induced relevant cellular and humoral immune responses. The project will include three specific aims. (1) Perform scale-up development of processes for GMP-compliant manufacture of a VRP vaccine expressing CMV pp65, IE1 and gB proteins. (2) Perform GMP manufacture of a VRP vaccine expressing these proteins. (3) Perform benchmark preclinical studies in preparation for an IND application. To achieve these aims, replicon plasmids containing CMV pp65, IE1 and gB genes and helper plasmids containing VEE capsid and glycoprotein genes will be prepared to GMP specifications. Purified RNA transcripts produced from the replicon and helper plasmids will be electroporated into certified Vero cells using various amounts and electroporation parameters to identify the optimal conditions for transfection efficiency and VRP yield. GMP lots will be manufactured based on experience with other VRP vaccine products. Preclinical toxicology and stability studies required for submission of IND application will be performed. Results of these studies will provide the basis for future clinical trials of a CMV VRP vaccine. PROPOSED COMMERCIAL APPLICATIONS: An effective CMV vaccine will prevent mental retardation and hearing loss associated with congenital CMV disease, thereby addressing an important unmet public health problem.